1. Field of the Invention
The present invention relates to a novel method and reagent useful for the measurement of lithium ions, in particular, lithium ions in blood and other physiological fluids.
2. Description of the Prior Art
The determination of lithium ion concentration has application in monitoring medical therapy. Specifically, lithium carbonate is frequently used in the treatment of manic depression and other psychiatric disorders, and the measurement of the lithium level in blood aids the physician in monitoring lithium drug therapy. Needless to say, a rapid, easy-to-perform method for determining the presence and concentration of a lithium ion in aqueous samples would greatly enhance such treatment.
Current methods for lithium measurement in clinical samples use flame photometry and only very recently ion-selective electrodes (ISE). An accurate determination of lithium in clinical samples by the ISE method is difficult and cumbersome, mainly due to interferences from sodium and other ions present in serum. To eliminate this problem a concurrent sodium measurement is required.
Lithium selective chromoionophores have been described in several publications. The first lithium specific chromogenic corand was described by G. E. Pacey et al., Synth. Commun. 11, 1981, 323-328. A chromogenic "crowned" phenol selective for lithium was described by T. Kaneda et al., Tetrahedron Lett. 22, 1981, 4407-4408. A chloroform solution of this compound gave a 160 nm bathochromic shift to a purple-red color upon contact with excess solid LiCl or LiClO.sub.4 (but not other salts) in the presence of pyridine. S. Ogawa et al., J. Am. Chem. Soc. 106, 1984, 5760-5762, reported a chromogenic compound which upon contact with LiCl in a methylene chloride solution turned from red to colorless. K. Sasaki et al., Anal. Chim. Acta 174, 1985, 141-149, reported another chromogenic corand which was used for the determination of lithium in an extraction system. Misumi et al., J. Am. Chem. Soc. 107, 1985, 4802-4803, synthesized a chromogenic spherand which acted as a lithium specific indicator for solid lithium salts of soft anions but was too weak a binder to strip water from Li(H.sub.2 O).sub.6.sup. +. A cation extraction study with a series of lithium-selective chromogenic corands was published by K. Kimura et al., J. Org. Chem. 52, 1987, 836-844. A. S. Attiyat et al., Microchem. J. 37, 1988, 114-121, described the spectrophotometric measurement of lithium in the presence of sodium using TMC-crown formazane. Another chromogenic spherand reported by D. J. Cram et al., J. Am. Chem. Soc. 110, 1988, 571-577 was capable of detecting lithium and sodium in an 80% dioxane-20% water system. Many of these prior art procedures require extraction-photometric procedures that are difficult to automate; sample pre-treatment and poor selectivity make them unattractive as alternatives to ISE or flame photometry. The relatively low therapeutic level of lithium in serum (0.5-1.5 mM) imposes very high constraints on selectivity over the high normal serum sodium concentration (135-150 mM). Ideally, the selectivity for lithium over sodium should be 1,500:1 in order to essentially eliminate any sodium interference.
The compounds of the present invention can generally be described as chromogenic (1.1.0) cryptands. Certain cryptands are known to have high selectivity for complexing with cations, and if coupled with chromophores, yield intensive color reactions that can be evaluated analytically. For example, Vogtle U.S. Pat. No. 4,367,072 describes a process for determining ions employing chromogenic cryptands. It is essentially based on ion-selective complexation between the ion to be determined and a complexing agent and measurement of the extinction change occurring during complexing. The complexing agent is bonded with a chromophore.
Klink, et al. European Patent Publication 85,320 discloses a potassium reagent and a procedure for determining potassium ions. The reagent contains a compound of general formula ##STR3## where n and m=0 or 1, X=N or COH and R=p-nitrophenylazo, 3-phenylisothiazolyl-5-azo, isothiazolyl-5-axo, thiazolyl-5-azo, 2,4,6-trinitrophenylazo, p-nitrostyryl, p-benzoquinonemonoimino and bis- (p-dimethylaminophenyl) hydroxy-methyl. The potassium ions are determined in a reaction medium consisting of water and at least one water-miscible organic solvent and in the presence of an organic base.
Both the Vogtle and Klink, et al. structures have larger size cavities designed for complexation with potassium ions. An ionophore for lithium, on the other hand, must have a much smaller and more pre-organized cavity in order to complex with the small=: lithium ion.
The fundamental difficulty in the design of lithium selective chromoionophores lies in the fact that lithium is the third smallest element (after hydrogen and helium) with ionic diameter of 1.20 (Na.sup.+ 1.90.ANG.; K.sup.+ 2.66.ANG.). The task then involves design and synthesis of an ionophore with a small cavity which is inflexible so as to exclude other ions from interaction for high selectivity and which possesses the binding sites preorganized complementarily for lithium complexation to achieve high sensitivity. The small cavity size puts severe strain on the cyclic structure making the synthesis of such ionophore extremely difficult. In general, the monocyclic crown ethers with small cavities, which are relatively easier to synthesize, have failed to meet the selectivity and sensitivity requirements because of lack of preorganization of the binding sites.
The major difference between the cryptands of the present invention and structures of the cryptands reported earlier by Vogtle, et al. and Klink, et al. is the lack of oxygen atoms in the side arms linking the cyclic diamine moiety with the aromatic subunit. It is known to those skilled in the art that such structural modifications usually lead to the loss of binding power by the cryptand and are not expected to be beneficial. However, quite unexpectedly, we discovered that the cryptands of this invention, devoid of side-arm oxygen arms, are able to bind lithium exclusively and fully discriminate over sodium. The removal of these oxygens from the side arms enabled us to construct cryptands with cavities in which the lithium ion can be held tightly and very fittingly. In addition, examination of molecular models indicates that the loss of oxygen binding sites is well compensated by the gain in preorganization of the cryptand cavity.
Accordingly, it has been found that the chromogenic cryptands of the present invention demonstrate particular sensitivity to lithium ions. The chromogenic cryptand can be incorporated into a reagent adapted for use on automated clinical analyzers to determine the lithium concentration in physiological fluid samples such as blood.